11836170001 superasin thermo am2696 rnasin promega n2515  (Thermo Fisher)


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    Name:
    SUPERase• In RNase Inhibitor 20 U μL
    Description:
    SUPERase• In RNase Inhibitor is a protein based inhibitor of nonhuman origin that noncovalently binds and inhibits the most common and troublesome RNases including RNase A B C 1 and T1 Features of SUPERase• In RNase Inhibitor • Completely removes RNase contamination from glass and plastic surfaces• Excels at removing high levels of RNase contamination where similar products fail• Proven effective at removing high concentrations of dried on RNase A• Ideal for cleaning work surfaces pipettors and equipment that must be RNase freeSUPERase• In RNase Inhibitor can be used in any application where RNase contamination could be problematic Because it inhibits a broader range of RNases than traditional RNase inhibitors SUPERase• In is the most effective RNase inhibitor available providing a higher level of protection against degradation SUPERase• In does not interfere with other enzymes such as RNA polymerases reverse transcriptase or Taq DNA polymerase Additionally SUPERase• In is active up to 65°C and over a pH range of 5 5 to 8 5 If you are plannning to use this product with standard antibody purification methods please contact our technical support to discuss experimental design Unit definition SUPERase• In RNase Inhibitor at 1 U µL will block the degradation of 0 1 µg µL labeled RNA by 2 5 pg µL of RNase A 2 5 pg µL of RNase I and 0 0075 U µL of RNase T1 for 4 hours at 37°C in 20 mM Tris HCl pH 7 5 50 mM NaCl 1 mM EDTA Analysis is by denaturing PAGE SUPERase• In is currently the only ribonuclease inhibitor for which the unit activity is defined by such a functional assay
    Catalog Number:
    am2694
    Price:
    None
    Applications:
    General Real-Time PCR Reagents|In Vitro Transcription|PCR & Real-Time PCR|Real Time PCR (qPCR)|Reverse Transcription|Gene Expression Analysis & Genotyping
    Category:
    Proteins Enzymes Peptides
    Buy from Supplier


    Structured Review

    Thermo Fisher 11836170001 superasin thermo am2696 rnasin promega n2515
    SUPERase• In RNase Inhibitor is a protein based inhibitor of nonhuman origin that noncovalently binds and inhibits the most common and troublesome RNases including RNase A B C 1 and T1 Features of SUPERase• In RNase Inhibitor • Completely removes RNase contamination from glass and plastic surfaces• Excels at removing high levels of RNase contamination where similar products fail• Proven effective at removing high concentrations of dried on RNase A• Ideal for cleaning work surfaces pipettors and equipment that must be RNase freeSUPERase• In RNase Inhibitor can be used in any application where RNase contamination could be problematic Because it inhibits a broader range of RNases than traditional RNase inhibitors SUPERase• In is the most effective RNase inhibitor available providing a higher level of protection against degradation SUPERase• In does not interfere with other enzymes such as RNA polymerases reverse transcriptase or Taq DNA polymerase Additionally SUPERase• In is active up to 65°C and over a pH range of 5 5 to 8 5 If you are plannning to use this product with standard antibody purification methods please contact our technical support to discuss experimental design Unit definition SUPERase• In RNase Inhibitor at 1 U µL will block the degradation of 0 1 µg µL labeled RNA by 2 5 pg µL of RNase A 2 5 pg µL of RNase I and 0 0075 U µL of RNase T1 for 4 hours at 37°C in 20 mM Tris HCl pH 7 5 50 mM NaCl 1 mM EDTA Analysis is by denaturing PAGE SUPERase• In is currently the only ribonuclease inhibitor for which the unit activity is defined by such a functional assay
    https://www.bioz.com/result/11836170001 superasin thermo am2696 rnasin promega n2515/product/Thermo Fisher
    Average 90 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    11836170001 superasin thermo am2696 rnasin promega n2515 - by Bioz Stars, 2020-02
    90/100 stars

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    Related Articles

    Centrifugation:

    Article Title: Enhanced Replication of Mouse Adenovirus Type 1 following Virus-Induced Degradation of Protein Kinase R (PKR)
    Article Snippet: Cells were collected at 24 hpi by scraping in ice-cold PBS containing 100 µg/ml cycloheximide, pelleting, and resuspending in 500 µl lysis buffer (20 mM Tris-Cl, 150 mM NaCl, 15 mM MgCl2 , 8% glycerol, 20 IU/ml SUPERase•In [Thermo Fisher Scientific catalog no. AM2696], 80 IU/ml murine RNase inhibitor [New England BioLabs catalog no. M0314S], 0.1 mg/ml heparin [Sigma H3393-50], 0.1 mg/ml cycloheximide, 1 mM DTT, 1× protease inhibitor [protease inhibitor cocktail kit; Thermo Scientific catalog no. 78410], 20 IU/ml Turbo DNase [Thermo Fisher Scientific catalog no. AM2238], 1% Triton X-100 [Sigma T9284]). .. Cells were lysed by passage through a chilled 26-gauge needle 10 times, vortex mixing for 30 s, and then incubating on ice for 5 min. Lysates were cleared by centrifugation for 5 min at 14,000 × g at 4°C.

    Article Title: Novel insight into circular RNA HECTD1 in astrocyte activation via autophagy by targeting MIR142-TIPARP: implications for cerebral ischemic stroke
    Article Snippet: .. On the following day, the cells were transfected with 3ʹ-biotinylated MIR142 or MIR142 -mutation at a final concentration of 50 nM for 36 h. The cells were then washed with PBS, briefly vortexed, and incubated in lysis buffer (20 mM Tris, pH 7.5, 200 mM NaCl, 2.5 mM MgCl2 , 0.05% Igepal [Sigma-Aldrich, 18,896], 60 U/ml Superase-In [Invitrogen, AM2694], 1 mM DTT [Sigma-Aldrich, 43,816], and protease inhibitors [Biotool, ]) on ice for 10 min. Lysates were precleared by centrifugation, and 50-μl aliquots of the samples were prepared for input. .. The remaining lysates were incubated with M-280 Streptavidin magnetic beads (Invitrogen, 11205D).

    Article Title: Condensin controls recruitment of RNA polymerase II to achieve nematode X-chromosome dosage compensation
    Article Snippet: After washing twice with M9 buffer, animals were washed with cold nuclear isolation buffer (250 mM sucrose, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl2 , 1 mM EGTA, 0.25% NP-40, 1 mM DTT, protease inhibitors, 4 U/ml SUPERaseIn [AM2696; Ambion, Grand Island, NY]). .. Douncing and collection of nuclei was performed for up to six rounds as follows: dounce with 10X pestle A, 10X pestle B, 5 min centrifugation at 100×g , removal of nuclei-containing supernatant, and addition of an equal volume of nuclear isolation buffer to the pellet.

    Article Title: Ras Suppresses TXNIP Expression by Restricting Ribosome Translocation
    Article Snippet: The cell pellet was collected and lysed in polysome lysis buffer (15 mM Tris [pH 7.4], 15 mM MgCl2 , 100 mM KCl, 1% Triton X-100, 1 mM DTT, 100 μg/ml CHX, 500 U/ml SuperaseIn [Invitrogen; no. AM2696]) containing protease inhibitors (1 mM PMSF, 2.5 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin) and phosphatase inhibitors (phosphatase inhibitor cocktails 1 and 2; Sigma; no. P2850 and P5726). .. The cell lysate was loaded onto the top of a sucrose gradient and centrifuged at 47,000 rpm for 1 h at 4°C in a SW55 Ti rotor (Beckman; no. 342194), and centrifugation was stopped with no brake.

    Mass Spectrometry:

    Article Title: Identification of a transporter complex responsible for the cytosolic entry of nitrogen-containing bisphosphonates
    Article Snippet: Reagents were obtained from the following sources: antibodies against LAMP2 (mouse, sc-18822), Ran BP3 (mouse, sc-373678) and Rap 1A (goat, sc-1482) were from Santa Cruz Biotechnology (Dallas, Texas); antibodies against GAPDH (rabbit, 2118), V5-tag (rabbit, 13202) and EEA1 (rabbit, 2411) were from Cell Signaling Technology (Danvers, Massachusetts); antibodies against Na+ /K+ -ATPase (mouse, ab7671) and Lamin B1 (rabbit, ab16048) were from Abcam (Cambridge, Massachusetts); antibodies against V5-tag (mouse, R960-25) and HDJ2 (mouse, MS-225-P0) were from ThermoFisher Scientific (Waltham, Massachusetts); antibodies against Caveolin-1 (rabbit, C3237) and HA-tag (rat, 11867423001) were from Sigma-Aldrich (Burlington, Massachusetts); antibody against EEA1 (mouse, 610456) was from BD Biosciences (San Jose, California); antibody against LMAP1 (mouse, H4A3) was from DSYB (Developmental Studies Hybridoma Bank, Iowa City, Iowa); antibody against Ubiquitin (mouse, 05–944) was from Millipore-Sigma (Burlington, Massachusetts); alendronate (A4978), lovastatin (PHR1285), zoledronate (SML0223), ibandronate (I5784), etidronate (P5248), tiludronate (T4580), chloroquine (C6628), puromycin (P8833), polybrene (H9268), anti-HA (A2095) and anti-V5 (A7345) agarose affinity matrix, poly-L-lysine solution (P4707), fibronectin solution (F0895), Triton X-100 (T8787), saponin (47036), Bovine Serum Albumin (BSA, A9647) and complete protease inhibitor cocktail (11836170001) were from Sigma-Aldrich; HRP-conjugated anti-rat secondary antibody (31470), Alexa 488 and Alexa 647-conjugated secondary antibodies ( , , and ), 0.1 µm TetraSpeck microspheres (T7279), SlowFade Diamond mounting medium , Halt protease-phosphatase inhibitor cocktail (78443), BCA protein assay kit (23225), SuperSignal west femto substrate (34095), TOPO TA cloning kit (450030), TRIzol and TRIzol LS reagents (15596018 and 10296028), SuperScript IV (18090050), RNase-free Turbo DNase (AM2238), SUPERase. .. In RNase Inhibitor (AM2694), SYBR Green qPCR master mix , Hygromycin B (10687010), DMEM (11965118), RPMI (11875093), Fetal Bovine Serum (FBS, 16000044) and Lipofectamine 3000 reagent (L3000008) were from ThermoFisher Scientific; HRP-conjugated anti-mouse and anti-rabbit secondary antibodies (1706515 and 1706516) were from Bio-Rad (Hercules, California); PNGase F (P0704) and Endo Hf (P0703) were from New England Biolabs (NEB, Ipswich, Massachusetts); cell line Nucleofector kit V (VACA-1003) was from Lonza (Walkersville, Maryland); CellTiter-Glo kit (G7572) was from Promega (Fitchburg, Wisconsin); mouse RANK ligand (RANKL, 462-TEC-010) was from R and D systems (Minneapolis, Minnesota); IMDM (30–2005) was from ATCC (American Type Culture Collection, Manassas, Virginia); digitonin (300410) was from Millipore-Sigma; QuickExtract DNA extraction solution (QE0905T) was from Epicentre (Madison, Wisconsin); tritium-labeled alendronate (MT-1727) was from Moravek (Brea, California); Alexa flour 647 labeled zoledronate (AF647-ZOL) was from BioVinc (Pasadena, California); Janelia flour 549 was a gift from Luke Lavis; pSpCas9(BB)−2A-GFP (PX458) was a gift from Feng Zhang (Addgene plasmid # 48138); AAVS1-Puro-PGK1 −3 × FLAG TwinStrep was a gift from Yannick Doyon (Addgene plasmid # 68375); pLenti PGK Hygro DEST (w530-1) was a gift from Eric Campeau and Paul Kaufman (Addgene plasmid # 19066); psPAX2 and pMD2.G were gifts from Didier Trono (Addgene plasmid # 12260 and # 12259).

    TA Cloning:

    Article Title: Identification of a transporter complex responsible for the cytosolic entry of nitrogen-containing bisphosphonates
    Article Snippet: Reagents were obtained from the following sources: antibodies against LAMP2 (mouse, sc-18822), Ran BP3 (mouse, sc-373678) and Rap 1A (goat, sc-1482) were from Santa Cruz Biotechnology (Dallas, Texas); antibodies against GAPDH (rabbit, 2118), V5-tag (rabbit, 13202) and EEA1 (rabbit, 2411) were from Cell Signaling Technology (Danvers, Massachusetts); antibodies against Na+ /K+ -ATPase (mouse, ab7671) and Lamin B1 (rabbit, ab16048) were from Abcam (Cambridge, Massachusetts); antibodies against V5-tag (mouse, R960-25) and HDJ2 (mouse, MS-225-P0) were from ThermoFisher Scientific (Waltham, Massachusetts); antibodies against Caveolin-1 (rabbit, C3237) and HA-tag (rat, 11867423001) were from Sigma-Aldrich (Burlington, Massachusetts); antibody against EEA1 (mouse, 610456) was from BD Biosciences (San Jose, California); antibody against LMAP1 (mouse, H4A3) was from DSYB (Developmental Studies Hybridoma Bank, Iowa City, Iowa); antibody against Ubiquitin (mouse, 05–944) was from Millipore-Sigma (Burlington, Massachusetts); alendronate (A4978), lovastatin (PHR1285), zoledronate (SML0223), ibandronate (I5784), etidronate (P5248), tiludronate (T4580), chloroquine (C6628), puromycin (P8833), polybrene (H9268), anti-HA (A2095) and anti-V5 (A7345) agarose affinity matrix, poly-L-lysine solution (P4707), fibronectin solution (F0895), Triton X-100 (T8787), saponin (47036), Bovine Serum Albumin (BSA, A9647) and complete protease inhibitor cocktail (11836170001) were from Sigma-Aldrich; HRP-conjugated anti-rat secondary antibody (31470), Alexa 488 and Alexa 647-conjugated secondary antibodies ( , , and ), 0.1 µm TetraSpeck microspheres (T7279), SlowFade Diamond mounting medium , Halt protease-phosphatase inhibitor cocktail (78443), BCA protein assay kit (23225), SuperSignal west femto substrate (34095), TOPO TA cloning kit (450030), TRIzol and TRIzol LS reagents (15596018 and 10296028), SuperScript IV (18090050), RNase-free Turbo DNase (AM2238), SUPERase. .. In RNase Inhibitor (AM2694), SYBR Green qPCR master mix , Hygromycin B (10687010), DMEM (11965118), RPMI (11875093), Fetal Bovine Serum (FBS, 16000044) and Lipofectamine 3000 reagent (L3000008) were from ThermoFisher Scientific; HRP-conjugated anti-mouse and anti-rabbit secondary antibodies (1706515 and 1706516) were from Bio-Rad (Hercules, California); PNGase F (P0704) and Endo Hf (P0703) were from New England Biolabs (NEB, Ipswich, Massachusetts); cell line Nucleofector kit V (VACA-1003) was from Lonza (Walkersville, Maryland); CellTiter-Glo kit (G7572) was from Promega (Fitchburg, Wisconsin); mouse RANK ligand (RANKL, 462-TEC-010) was from R and D systems (Minneapolis, Minnesota); IMDM (30–2005) was from ATCC (American Type Culture Collection, Manassas, Virginia); digitonin (300410) was from Millipore-Sigma; QuickExtract DNA extraction solution (QE0905T) was from Epicentre (Madison, Wisconsin); tritium-labeled alendronate (MT-1727) was from Moravek (Brea, California); Alexa flour 647 labeled zoledronate (AF647-ZOL) was from BioVinc (Pasadena, California); Janelia flour 549 was a gift from Luke Lavis; pSpCas9(BB)−2A-GFP (PX458) was a gift from Feng Zhang (Addgene plasmid # 48138); AAVS1-Puro-PGK1 −3 × FLAG TwinStrep was a gift from Yannick Doyon (Addgene plasmid # 68375); pLenti PGK Hygro DEST (w530-1) was a gift from Eric Campeau and Paul Kaufman (Addgene plasmid # 19066); psPAX2 and pMD2.G were gifts from Didier Trono (Addgene plasmid # 12260 and # 12259).

    Construct:

    Article Title: Selective 2′-Hydroxyl Acylation analyzed by Protection from Exoribonuclease (RNase-detected SHAPE): direct analysis of covalent adducts and of nucleotide flexibility in RNA
    Article Snippet: .. M. genitalium RNase R, 4.5 mg/mL, conveniently obtained by affinity purification using a C-terminal (His)6 -tagged expression construct (see Supporting Protocol) Thiamine pyrophosphate (TPP) (Sigma-Aldrich, cat. no. C8754) Boric acid (Sigma-Aldrich, cat. no. B7901) SUPERase-In (Ambion, cat. no. AM2694) Alkaline phosphatase (Roche, cat. no. 1097075) T4 polynucleotide kinase (PNK) (New England Biolabs, cat. no. M0201S) 10× PNK buffer (New England Biolabs, cat. no. M0201S) γ-[32 P]-ATP (6 × 106 Ci/mol, 10 Ci/L, Perkin Elmer, cat. no. BLU502Z) Reagents for high-resolution polyacrylamide gel electrophoresis, including 29:1 acrylamide:bisacrylamide, 7 M urea, 1×TBE ..

    Real-time Polymerase Chain Reaction:

    Article Title: Identification of a transporter complex responsible for the cytosolic entry of nitrogen-containing bisphosphonates
    Article Snippet: .. In RNase Inhibitor (AM2694), SYBR Green qPCR master mix , Hygromycin B (10687010), DMEM (11965118), RPMI (11875093), Fetal Bovine Serum (FBS, 16000044) and Lipofectamine 3000 reagent (L3000008) were from ThermoFisher Scientific; HRP-conjugated anti-mouse and anti-rabbit secondary antibodies (1706515 and 1706516) were from Bio-Rad (Hercules, California); PNGase F (P0704) and Endo Hf (P0703) were from New England Biolabs (NEB, Ipswich, Massachusetts); cell line Nucleofector kit V (VACA-1003) was from Lonza (Walkersville, Maryland); CellTiter-Glo kit (G7572) was from Promega (Fitchburg, Wisconsin); mouse RANK ligand (RANKL, 462-TEC-010) was from R and D systems (Minneapolis, Minnesota); IMDM (30–2005) was from ATCC (American Type Culture Collection, Manassas, Virginia); digitonin (300410) was from Millipore-Sigma; QuickExtract DNA extraction solution (QE0905T) was from Epicentre (Madison, Wisconsin); tritium-labeled alendronate (MT-1727) was from Moravek (Brea, California); Alexa flour 647 labeled zoledronate (AF647-ZOL) was from BioVinc (Pasadena, California); Janelia flour 549 was a gift from Luke Lavis; pSpCas9(BB)−2A-GFP (PX458) was a gift from Feng Zhang (Addgene plasmid # 48138); AAVS1-Puro-PGK1 −3 × FLAG TwinStrep was a gift from Yannick Doyon (Addgene plasmid # 68375); pLenti PGK Hygro DEST (w530-1) was a gift from Eric Campeau and Paul Kaufman (Addgene plasmid # 19066); psPAX2 and pMD2.G were gifts from Didier Trono (Addgene plasmid # 12260 and # 12259). .. K562 human myeloid leukemia cells, Human Embryo Kidney (HEK) 293 T cells and RAW 264.7 murine macrophage-like cells were obtained from ATCC.

    Microarray:

    Article Title: Using intracellular markers to identify a novel set of surface markers for live cell purification from a heterogeneous hIPSC culture
    Article Snippet: Paragraph title: MARIS and Microarray ... The washed pellet was then resuspended in perm/block buffer (0.5% saponin, 1% BSA, 200 U/mL Superase.In RNAse inhibitor (Life Technologies cat# AM2696), in RNAse-free PBS), in the same volume as used for fixation, with primary antibody and incubated at room temperature for two hours with occasional mixing.

    Incubation:

    Article Title: Nucleotide Resolution Comparison of Transcription of Human Cytomegalovirus and Host Genomes Reveals Universal Use of RNA Polymerase II Elongation Control Driven by Dissimilar Core Promoter Elements
    Article Snippet: After infection and treatment of cells, T150 flasks were removed from the incubator, and over 20 s, medium was discarded; phosphate-buffered saline (PBS) was added, quickly mixed, and discarded, and 10 ml lysis buffer (20 mM HEPES, pH 7.6, 300 mM sucrose, 1% IGEPAL CA-630, 1 mM spermine, 1 mM spermidine, 1 mM EDTA, 1 mM dithiothreitol [DTT], 0.004 U/μl SUPERase-In [Ambion AM2696], 0.1% isopropanol-saturated phenylmethylsulfonyl fluoride [PMSF], and cOmplete EDTA-free protease inhibitor cocktail [Roche 11873580001]) was introduced. .. After a brief incubation on ice, lysis was confirmed by phase-contrast microscopy.

    Article Title: Novel insight into circular RNA HECTD1 in astrocyte activation via autophagy by targeting MIR142-TIPARP: implications for cerebral ischemic stroke
    Article Snippet: .. On the following day, the cells were transfected with 3ʹ-biotinylated MIR142 or MIR142 -mutation at a final concentration of 50 nM for 36 h. The cells were then washed with PBS, briefly vortexed, and incubated in lysis buffer (20 mM Tris, pH 7.5, 200 mM NaCl, 2.5 mM MgCl2 , 0.05% Igepal [Sigma-Aldrich, 18,896], 60 U/ml Superase-In [Invitrogen, AM2694], 1 mM DTT [Sigma-Aldrich, 43,816], and protease inhibitors [Biotool, ]) on ice for 10 min. Lysates were precleared by centrifugation, and 50-μl aliquots of the samples were prepared for input. .. The remaining lysates were incubated with M-280 Streptavidin magnetic beads (Invitrogen, 11205D).

    Article Title: Using intracellular markers to identify a novel set of surface markers for live cell purification from a heterogeneous hIPSC culture
    Article Snippet: .. The washed pellet was then resuspended in perm/block buffer (0.5% saponin, 1% BSA, 200 U/mL Superase.In RNAse inhibitor (Life Technologies cat# AM2696), in RNAse-free PBS), in the same volume as used for fixation, with primary antibody and incubated at room temperature for two hours with occasional mixing. ..

    Article Title: EINCR1 is an EGF inducible lincRNA overexpressed in lung adenocarcinomas
    Article Snippet: .. Briefly, cells were washed with 1xPBS and then incubated for 10 min with 1 ml of RLN buffer (50 mM TrisHCl pH = 8.0, 140 mM NaCl, 1.5 mM MgCl2 , 0.5% (v/v) IGEPAL CA-630 and 2 U/ml SUPERaseIn—RNase inhibitor, Invitrogen AM2696). .. Subsequently, the lysate was centrifuged at 1,000 g for 5 min at 4°C, the pellet was collected, resuspended in 1 ml of RLN buffer, incubated for a further 5 min on ice and centrifuged at 1 000 g for 5 min at 4°C.

    Article Title: Ras Suppresses TXNIP Expression by Restricting Ribosome Translocation
    Article Snippet: Cells were washed and incubated with ice-cold phosphate-buffered saline (PBS) containing 100 μg/ml CHX for 2 min. .. The cell pellet was collected and lysed in polysome lysis buffer (15 mM Tris [pH 7.4], 15 mM MgCl2 , 100 mM KCl, 1% Triton X-100, 1 mM DTT, 100 μg/ml CHX, 500 U/ml SuperaseIn [Invitrogen; no. AM2696]) containing protease inhibitors (1 mM PMSF, 2.5 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin) and phosphatase inhibitors (phosphatase inhibitor cocktails 1 and 2; Sigma; no. P2850 and P5726).

    Formalin-fixed Paraffin-Embedded:

    Article Title: Species and cell-type properties of classically defined human and rodent neurons and glia
    Article Snippet: .. Other reagents Optiprep – Sigma D1556-250ML cOmplete mini protease inhibitors (EDTA free) – Roche distributed by Sigma 11836170001 Superasin – Thermo AM2696 RNasin – Promega N2515 16%formaldehyde solution – EMS 15710 BSA – Jackson 001-000-162 BSA RNase free – Thermo AM2618 DyeCycle Ruby – Thermo V10273 Kits Qiagen AllPrep FFPE – Cat #80234 Qiagen MinElute Reaction Cleanup – Cat #28206 Nugen Ovation RNAseq NEBNext Ultra II DNA Library Prep Kit for Illumina - E7645L NEBNext Multiplex Adapters for Illumina – E7335L (Set 1) and E7500L (Set 2) Qubit dsDNA High Sensitivity - Qubit RNA HS - Bioanalyzer Pico chips - 5067–1513 TapeStation D1000 - 5067–5582, ScreenTape; 5067–5583 TapeStation High Sensitivity D1000 – 5067–5584, ScreenTape; 5067–5585 Equipment Dounce homogenizer – 40 mL all glass homogenizer with large and small clearance pestles – Kimble Chase 885300–0040 Ultracentrifuge – Beckman Optima XE, Beckman SW41 Ti swinging rotor and buckets, Beckman Ultraclear 13.2 mL tubes (41121703) Refrigerated benchtop centrifuge similar to Eppendorf 5424R Nutrating Mixer FACS – BD FACSAria with 488, 561, 635/640 lasers Flow cytometry analyzer – BD LSR II with 488, 561, 640 lasers Software – Diva and FlowJo Agilent Bioanalyzer – with pico and nano chips for RNA Agilent TapeStation – D1000 and High Sensitivity D1000 screentapes and reagents Qubit Florometer Roche LightCycler 480 Covaris S220 Focused Ultrasonicator Dynamags from Thermo – 96 side magnet 12331D .. Mice were deeply anesthetized and transcardially perfused with phosphate buffered saline (PBS) followed by 4% formaldehyde (w/v) in PBS.

    Infection:

    Article Title: Nucleotide Resolution Comparison of Transcription of Human Cytomegalovirus and Host Genomes Reveals Universal Use of RNA Polymerase II Elongation Control Driven by Dissimilar Core Promoter Elements
    Article Snippet: .. After infection and treatment of cells, T150 flasks were removed from the incubator, and over 20 s, medium was discarded; phosphate-buffered saline (PBS) was added, quickly mixed, and discarded, and 10 ml lysis buffer (20 mM HEPES, pH 7.6, 300 mM sucrose, 1% IGEPAL CA-630, 1 mM spermine, 1 mM spermidine, 1 mM EDTA, 1 mM dithiothreitol [DTT], 0.004 U/μl SUPERase-In [Ambion AM2696], 0.1% isopropanol-saturated phenylmethylsulfonyl fluoride [PMSF], and cOmplete EDTA-free protease inhibitor cocktail [Roche 11873580001]) was introduced. .. After a brief incubation on ice, lysis was confirmed by phase-contrast microscopy.

    Article Title: Enhanced Replication of Mouse Adenovirus Type 1 following Virus-Induced Degradation of Protein Kinase R (PKR)
    Article Snippet: The next day, the cells were infected with MAV-1 at an MOI of 2. .. Cells were collected at 24 hpi by scraping in ice-cold PBS containing 100 µg/ml cycloheximide, pelleting, and resuspending in 500 µl lysis buffer (20 mM Tris-Cl, 150 mM NaCl, 15 mM MgCl2 , 8% glycerol, 20 IU/ml SUPERase•In [Thermo Fisher Scientific catalog no. AM2696], 80 IU/ml murine RNase inhibitor [New England BioLabs catalog no. M0314S], 0.1 mg/ml heparin [Sigma H3393-50], 0.1 mg/ml cycloheximide, 1 mM DTT, 1× protease inhibitor [protease inhibitor cocktail kit; Thermo Scientific catalog no. 78410], 20 IU/ml Turbo DNase [Thermo Fisher Scientific catalog no. AM2238], 1% Triton X-100 [Sigma T9284]).

    Expressing:

    Article Title: Selective 2′-Hydroxyl Acylation analyzed by Protection from Exoribonuclease (RNase-detected SHAPE): direct analysis of covalent adducts and of nucleotide flexibility in RNA
    Article Snippet: .. M. genitalium RNase R, 4.5 mg/mL, conveniently obtained by affinity purification using a C-terminal (His)6 -tagged expression construct (see Supporting Protocol) Thiamine pyrophosphate (TPP) (Sigma-Aldrich, cat. no. C8754) Boric acid (Sigma-Aldrich, cat. no. B7901) SUPERase-In (Ambion, cat. no. AM2694) Alkaline phosphatase (Roche, cat. no. 1097075) T4 polynucleotide kinase (PNK) (New England Biolabs, cat. no. M0201S) 10× PNK buffer (New England Biolabs, cat. no. M0201S) γ-[32 P]-ATP (6 × 106 Ci/mol, 10 Ci/L, Perkin Elmer, cat. no. BLU502Z) Reagents for high-resolution polyacrylamide gel electrophoresis, including 29:1 acrylamide:bisacrylamide, 7 M urea, 1×TBE ..

    Article Title: Combining NGN2 Programming with Developmental Patterning Generates Human Excitatory Neurons with NMDAR-Mediated Synaptic Transmission
    Article Snippet: Paragraph title: Single-Cell Expression Profiling and Transcriptomics ... For Fluidigm Biomark, cells were single-cell sorted into 5 μL of 10 mM Tris (pH 8.0), 0.1 mM EDTA supplemented with SUPERase•In (0.1 U/μL final, Ambion, AM2696), and 0.5% NP40 (Thermo Scientific, PI-28324).

    Article Title: Convergence of independent DISC1 mutations on impaired neurite growth via decreased UNC5D expression
    Article Snippet: .. Day 28 cultures were single-cell sorted for GFP expression (marking transduced neurons) into lysis buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA with 0.5% NP40 [Thermo Scientific PI-28324] and 100 U/ml SUPERase•In™ [Ambion AM2696]). ..

    BIA-KA:

    Article Title: Identification of a transporter complex responsible for the cytosolic entry of nitrogen-containing bisphosphonates
    Article Snippet: Reagents were obtained from the following sources: antibodies against LAMP2 (mouse, sc-18822), Ran BP3 (mouse, sc-373678) and Rap 1A (goat, sc-1482) were from Santa Cruz Biotechnology (Dallas, Texas); antibodies against GAPDH (rabbit, 2118), V5-tag (rabbit, 13202) and EEA1 (rabbit, 2411) were from Cell Signaling Technology (Danvers, Massachusetts); antibodies against Na+ /K+ -ATPase (mouse, ab7671) and Lamin B1 (rabbit, ab16048) were from Abcam (Cambridge, Massachusetts); antibodies against V5-tag (mouse, R960-25) and HDJ2 (mouse, MS-225-P0) were from ThermoFisher Scientific (Waltham, Massachusetts); antibodies against Caveolin-1 (rabbit, C3237) and HA-tag (rat, 11867423001) were from Sigma-Aldrich (Burlington, Massachusetts); antibody against EEA1 (mouse, 610456) was from BD Biosciences (San Jose, California); antibody against LMAP1 (mouse, H4A3) was from DSYB (Developmental Studies Hybridoma Bank, Iowa City, Iowa); antibody against Ubiquitin (mouse, 05–944) was from Millipore-Sigma (Burlington, Massachusetts); alendronate (A4978), lovastatin (PHR1285), zoledronate (SML0223), ibandronate (I5784), etidronate (P5248), tiludronate (T4580), chloroquine (C6628), puromycin (P8833), polybrene (H9268), anti-HA (A2095) and anti-V5 (A7345) agarose affinity matrix, poly-L-lysine solution (P4707), fibronectin solution (F0895), Triton X-100 (T8787), saponin (47036), Bovine Serum Albumin (BSA, A9647) and complete protease inhibitor cocktail (11836170001) were from Sigma-Aldrich; HRP-conjugated anti-rat secondary antibody (31470), Alexa 488 and Alexa 647-conjugated secondary antibodies ( , , and ), 0.1 µm TetraSpeck microspheres (T7279), SlowFade Diamond mounting medium , Halt protease-phosphatase inhibitor cocktail (78443), BCA protein assay kit (23225), SuperSignal west femto substrate (34095), TOPO TA cloning kit (450030), TRIzol and TRIzol LS reagents (15596018 and 10296028), SuperScript IV (18090050), RNase-free Turbo DNase (AM2238), SUPERase. .. In RNase Inhibitor (AM2694), SYBR Green qPCR master mix , Hygromycin B (10687010), DMEM (11965118), RPMI (11875093), Fetal Bovine Serum (FBS, 16000044) and Lipofectamine 3000 reagent (L3000008) were from ThermoFisher Scientific; HRP-conjugated anti-mouse and anti-rabbit secondary antibodies (1706515 and 1706516) were from Bio-Rad (Hercules, California); PNGase F (P0704) and Endo Hf (P0703) were from New England Biolabs (NEB, Ipswich, Massachusetts); cell line Nucleofector kit V (VACA-1003) was from Lonza (Walkersville, Maryland); CellTiter-Glo kit (G7572) was from Promega (Fitchburg, Wisconsin); mouse RANK ligand (RANKL, 462-TEC-010) was from R and D systems (Minneapolis, Minnesota); IMDM (30–2005) was from ATCC (American Type Culture Collection, Manassas, Virginia); digitonin (300410) was from Millipore-Sigma; QuickExtract DNA extraction solution (QE0905T) was from Epicentre (Madison, Wisconsin); tritium-labeled alendronate (MT-1727) was from Moravek (Brea, California); Alexa flour 647 labeled zoledronate (AF647-ZOL) was from BioVinc (Pasadena, California); Janelia flour 549 was a gift from Luke Lavis; pSpCas9(BB)−2A-GFP (PX458) was a gift from Feng Zhang (Addgene plasmid # 48138); AAVS1-Puro-PGK1 −3 × FLAG TwinStrep was a gift from Yannick Doyon (Addgene plasmid # 68375); pLenti PGK Hygro DEST (w530-1) was a gift from Eric Campeau and Paul Kaufman (Addgene plasmid # 19066); psPAX2 and pMD2.G were gifts from Didier Trono (Addgene plasmid # 12260 and # 12259).

    Modification:

    Article Title: Selective 2′-Hydroxyl Acylation analyzed by Protection from Exoribonuclease (RNase-detected SHAPE): direct analysis of covalent adducts and of nucleotide flexibility in RNA
    Article Snippet: RNA for modification and RNase R digestion can be stored in aliquots to reduce RNase contamination. .. M. genitalium RNase R, 4.5 mg/mL, conveniently obtained by affinity purification using a C-terminal (His)6 -tagged expression construct (see Supporting Protocol) Thiamine pyrophosphate (TPP) (Sigma-Aldrich, cat. no. C8754) Boric acid (Sigma-Aldrich, cat. no. B7901) SUPERase-In (Ambion, cat. no. AM2694) Alkaline phosphatase (Roche, cat. no. 1097075) T4 polynucleotide kinase (PNK) (New England Biolabs, cat. no. M0201S) 10× PNK buffer (New England Biolabs, cat. no. M0201S) γ-[32 P]-ATP (6 × 106 Ci/mol, 10 Ci/L, Perkin Elmer, cat. no. BLU502Z) Reagents for high-resolution polyacrylamide gel electrophoresis, including 29:1 acrylamide:bisacrylamide, 7 M urea, 1×TBE

    Article Title: Using intracellular markers to identify a novel set of surface markers for live cell purification from a heterogeneous hIPSC culture
    Article Snippet: MARIS and Microarray Whole RNA was extracted from LMX1+ FOXA2+ and LMX1- FOXA2- day 14 18a cells using a modification of the MARIS method, as previously established by our laboratory , . .. The washed pellet was then resuspended in perm/block buffer (0.5% saponin, 1% BSA, 200 U/mL Superase.In RNAse inhibitor (Life Technologies cat# AM2696), in RNAse-free PBS), in the same volume as used for fixation, with primary antibody and incubated at room temperature for two hours with occasional mixing.

    Article Title: Convergence of independent DISC1 mutations on impaired neurite growth via decreased UNC5D expression
    Article Snippet: Single-cell qRT-PCR For single cell experiments, the NGN2 protocol was modified to include dual SMAD inhibition and WNT inhibition during a critical window of differentiation . .. Day 28 cultures were single-cell sorted for GFP expression (marking transduced neurons) into lysis buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA with 0.5% NP40 [Thermo Scientific PI-28324] and 100 U/ml SUPERase•In™ [Ambion AM2696]).

    Inhibition:

    Article Title: Convergence of independent DISC1 mutations on impaired neurite growth via decreased UNC5D expression
    Article Snippet: Single-cell qRT-PCR For single cell experiments, the NGN2 protocol was modified to include dual SMAD inhibition and WNT inhibition during a critical window of differentiation . .. Day 28 cultures were single-cell sorted for GFP expression (marking transduced neurons) into lysis buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA with 0.5% NP40 [Thermo Scientific PI-28324] and 100 U/ml SUPERase•In™ [Ambion AM2696]).

    Transfection:

    Article Title: Novel insight into circular RNA HECTD1 in astrocyte activation via autophagy by targeting MIR142-TIPARP: implications for cerebral ischemic stroke
    Article Snippet: .. On the following day, the cells were transfected with 3ʹ-biotinylated MIR142 or MIR142 -mutation at a final concentration of 50 nM for 36 h. The cells were then washed with PBS, briefly vortexed, and incubated in lysis buffer (20 mM Tris, pH 7.5, 200 mM NaCl, 2.5 mM MgCl2 , 0.05% Igepal [Sigma-Aldrich, 18,896], 60 U/ml Superase-In [Invitrogen, AM2694], 1 mM DTT [Sigma-Aldrich, 43,816], and protease inhibitors [Biotool, ]) on ice for 10 min. Lysates were precleared by centrifugation, and 50-μl aliquots of the samples were prepared for input. .. The remaining lysates were incubated with M-280 Streptavidin magnetic beads (Invitrogen, 11205D).

    Protease Inhibitor:

    Article Title: Motor neuron-derived microRNAs cause astrocyte dysfunction in amyotrophic lateral sclerosis
    Article Snippet: .. The beads were washed thoroughly with buffer (10 mM KCl, 1.5 mM MgCl2 , 1 M NaCl, 10 mM Tris-HCl pH 7.5, 5 mM DTT, 0.5% Sigma-IGEPAL, 60 U/ml SUPERase In™) (Ambion; AM2694) and 1× complete mini protease inhibitor (Roche; 11836170001) before applying 300 μl of CSF with end-over rotating for 30 min at 4°C. ..

    Article Title: Nucleotide Resolution Comparison of Transcription of Human Cytomegalovirus and Host Genomes Reveals Universal Use of RNA Polymerase II Elongation Control Driven by Dissimilar Core Promoter Elements
    Article Snippet: .. After infection and treatment of cells, T150 flasks were removed from the incubator, and over 20 s, medium was discarded; phosphate-buffered saline (PBS) was added, quickly mixed, and discarded, and 10 ml lysis buffer (20 mM HEPES, pH 7.6, 300 mM sucrose, 1% IGEPAL CA-630, 1 mM spermine, 1 mM spermidine, 1 mM EDTA, 1 mM dithiothreitol [DTT], 0.004 U/μl SUPERase-In [Ambion AM2696], 0.1% isopropanol-saturated phenylmethylsulfonyl fluoride [PMSF], and cOmplete EDTA-free protease inhibitor cocktail [Roche 11873580001]) was introduced. .. After a brief incubation on ice, lysis was confirmed by phase-contrast microscopy.

    Article Title: Chromatin run-on and sequencing maps the transcriptional regulatory landscape of glioblastoma multiforme
    Article Snippet: .. For chromatin (ChRO) isolation from cultured cells or tissue we added 1 ml of 1× NUN Buffer (0.3 M NaCl, 1M Urea, 1% NP-40, 20 mM HEPES, pH 7.5, 7.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 20 units/ml RNase Inhibitor (Life Technologies # AM2694), 1× Protease Inhibitor Cocktail (Roche # 11 873 580 001)). ..

    Article Title: Enhanced Replication of Mouse Adenovirus Type 1 following Virus-Induced Degradation of Protein Kinase R (PKR)
    Article Snippet: .. Cells were collected at 24 hpi by scraping in ice-cold PBS containing 100 µg/ml cycloheximide, pelleting, and resuspending in 500 µl lysis buffer (20 mM Tris-Cl, 150 mM NaCl, 15 mM MgCl2 , 8% glycerol, 20 IU/ml SUPERase•In [Thermo Fisher Scientific catalog no. AM2696], 80 IU/ml murine RNase inhibitor [New England BioLabs catalog no. M0314S], 0.1 mg/ml heparin [Sigma H3393-50], 0.1 mg/ml cycloheximide, 1 mM DTT, 1× protease inhibitor [protease inhibitor cocktail kit; Thermo Scientific catalog no. 78410], 20 IU/ml Turbo DNase [Thermo Fisher Scientific catalog no. AM2238], 1% Triton X-100 [Sigma T9284]). .. Cells were lysed by passage through a chilled 26-gauge needle 10 times, vortex mixing for 30 s, and then incubating on ice for 5 min. Lysates were cleared by centrifugation for 5 min at 14,000 × g at 4°C.

    Article Title: Identification of a transporter complex responsible for the cytosolic entry of nitrogen-containing bisphosphonates
    Article Snippet: Reagents were obtained from the following sources: antibodies against LAMP2 (mouse, sc-18822), Ran BP3 (mouse, sc-373678) and Rap 1A (goat, sc-1482) were from Santa Cruz Biotechnology (Dallas, Texas); antibodies against GAPDH (rabbit, 2118), V5-tag (rabbit, 13202) and EEA1 (rabbit, 2411) were from Cell Signaling Technology (Danvers, Massachusetts); antibodies against Na+ /K+ -ATPase (mouse, ab7671) and Lamin B1 (rabbit, ab16048) were from Abcam (Cambridge, Massachusetts); antibodies against V5-tag (mouse, R960-25) and HDJ2 (mouse, MS-225-P0) were from ThermoFisher Scientific (Waltham, Massachusetts); antibodies against Caveolin-1 (rabbit, C3237) and HA-tag (rat, 11867423001) were from Sigma-Aldrich (Burlington, Massachusetts); antibody against EEA1 (mouse, 610456) was from BD Biosciences (San Jose, California); antibody against LMAP1 (mouse, H4A3) was from DSYB (Developmental Studies Hybridoma Bank, Iowa City, Iowa); antibody against Ubiquitin (mouse, 05–944) was from Millipore-Sigma (Burlington, Massachusetts); alendronate (A4978), lovastatin (PHR1285), zoledronate (SML0223), ibandronate (I5784), etidronate (P5248), tiludronate (T4580), chloroquine (C6628), puromycin (P8833), polybrene (H9268), anti-HA (A2095) and anti-V5 (A7345) agarose affinity matrix, poly-L-lysine solution (P4707), fibronectin solution (F0895), Triton X-100 (T8787), saponin (47036), Bovine Serum Albumin (BSA, A9647) and complete protease inhibitor cocktail (11836170001) were from Sigma-Aldrich; HRP-conjugated anti-rat secondary antibody (31470), Alexa 488 and Alexa 647-conjugated secondary antibodies ( , , and ), 0.1 µm TetraSpeck microspheres (T7279), SlowFade Diamond mounting medium , Halt protease-phosphatase inhibitor cocktail (78443), BCA protein assay kit (23225), SuperSignal west femto substrate (34095), TOPO TA cloning kit (450030), TRIzol and TRIzol LS reagents (15596018 and 10296028), SuperScript IV (18090050), RNase-free Turbo DNase (AM2238), SUPERase. .. In RNase Inhibitor (AM2694), SYBR Green qPCR master mix , Hygromycin B (10687010), DMEM (11965118), RPMI (11875093), Fetal Bovine Serum (FBS, 16000044) and Lipofectamine 3000 reagent (L3000008) were from ThermoFisher Scientific; HRP-conjugated anti-mouse and anti-rabbit secondary antibodies (1706515 and 1706516) were from Bio-Rad (Hercules, California); PNGase F (P0704) and Endo Hf (P0703) were from New England Biolabs (NEB, Ipswich, Massachusetts); cell line Nucleofector kit V (VACA-1003) was from Lonza (Walkersville, Maryland); CellTiter-Glo kit (G7572) was from Promega (Fitchburg, Wisconsin); mouse RANK ligand (RANKL, 462-TEC-010) was from R and D systems (Minneapolis, Minnesota); IMDM (30–2005) was from ATCC (American Type Culture Collection, Manassas, Virginia); digitonin (300410) was from Millipore-Sigma; QuickExtract DNA extraction solution (QE0905T) was from Epicentre (Madison, Wisconsin); tritium-labeled alendronate (MT-1727) was from Moravek (Brea, California); Alexa flour 647 labeled zoledronate (AF647-ZOL) was from BioVinc (Pasadena, California); Janelia flour 549 was a gift from Luke Lavis; pSpCas9(BB)−2A-GFP (PX458) was a gift from Feng Zhang (Addgene plasmid # 48138); AAVS1-Puro-PGK1 −3 × FLAG TwinStrep was a gift from Yannick Doyon (Addgene plasmid # 68375); pLenti PGK Hygro DEST (w530-1) was a gift from Eric Campeau and Paul Kaufman (Addgene plasmid # 19066); psPAX2 and pMD2.G were gifts from Didier Trono (Addgene plasmid # 12260 and # 12259).

    Cell Culture:

    Article Title: Chromatin run-on and sequencing maps the transcriptional regulatory landscape of glioblastoma multiforme
    Article Snippet: .. For chromatin (ChRO) isolation from cultured cells or tissue we added 1 ml of 1× NUN Buffer (0.3 M NaCl, 1M Urea, 1% NP-40, 20 mM HEPES, pH 7.5, 7.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 20 units/ml RNase Inhibitor (Life Technologies # AM2694), 1× Protease Inhibitor Cocktail (Roche # 11 873 580 001)). ..

    Labeling:

    Article Title: Identification of a transporter complex responsible for the cytosolic entry of nitrogen-containing bisphosphonates
    Article Snippet: .. In RNase Inhibitor (AM2694), SYBR Green qPCR master mix , Hygromycin B (10687010), DMEM (11965118), RPMI (11875093), Fetal Bovine Serum (FBS, 16000044) and Lipofectamine 3000 reagent (L3000008) were from ThermoFisher Scientific; HRP-conjugated anti-mouse and anti-rabbit secondary antibodies (1706515 and 1706516) were from Bio-Rad (Hercules, California); PNGase F (P0704) and Endo Hf (P0703) were from New England Biolabs (NEB, Ipswich, Massachusetts); cell line Nucleofector kit V (VACA-1003) was from Lonza (Walkersville, Maryland); CellTiter-Glo kit (G7572) was from Promega (Fitchburg, Wisconsin); mouse RANK ligand (RANKL, 462-TEC-010) was from R and D systems (Minneapolis, Minnesota); IMDM (30–2005) was from ATCC (American Type Culture Collection, Manassas, Virginia); digitonin (300410) was from Millipore-Sigma; QuickExtract DNA extraction solution (QE0905T) was from Epicentre (Madison, Wisconsin); tritium-labeled alendronate (MT-1727) was from Moravek (Brea, California); Alexa flour 647 labeled zoledronate (AF647-ZOL) was from BioVinc (Pasadena, California); Janelia flour 549 was a gift from Luke Lavis; pSpCas9(BB)−2A-GFP (PX458) was a gift from Feng Zhang (Addgene plasmid # 48138); AAVS1-Puro-PGK1 −3 × FLAG TwinStrep was a gift from Yannick Doyon (Addgene plasmid # 68375); pLenti PGK Hygro DEST (w530-1) was a gift from Eric Campeau and Paul Kaufman (Addgene plasmid # 19066); psPAX2 and pMD2.G were gifts from Didier Trono (Addgene plasmid # 12260 and # 12259). .. K562 human myeloid leukemia cells, Human Embryo Kidney (HEK) 293 T cells and RAW 264.7 murine macrophage-like cells were obtained from ATCC.

    other:

    Article Title: High-Throughput Sequencing of RNA Isolated by Cross- Linking and Immunoprecipitation (HITS-CLIP) to Determine Sites of Binding of CstF-64 on Nascent RNAs
    Article Snippet: The following enzymes and kits were used as recommended by the suppliers: SUPERase ⋅ In (AM2696, Life Technologies).

    Sequencing:

    Article Title: Species and cell-type properties of classically defined human and rodent neurons and glia
    Article Snippet: Submit for sequencing with TapeStation results. .. Other reagents Optiprep – Sigma D1556-250ML cOmplete mini protease inhibitors (EDTA free) – Roche distributed by Sigma 11836170001 Superasin – Thermo AM2696 RNasin – Promega N2515 16%formaldehyde solution – EMS 15710 BSA – Jackson 001-000-162 BSA RNase free – Thermo AM2618 DyeCycle Ruby – Thermo V10273 Kits Qiagen AllPrep FFPE – Cat #80234 Qiagen MinElute Reaction Cleanup – Cat #28206 Nugen Ovation RNAseq NEBNext Ultra II DNA Library Prep Kit for Illumina - E7645L NEBNext Multiplex Adapters for Illumina – E7335L (Set 1) and E7500L (Set 2) Qubit dsDNA High Sensitivity - Qubit RNA HS - Bioanalyzer Pico chips - 5067–1513 TapeStation D1000 - 5067–5582, ScreenTape; 5067–5583 TapeStation High Sensitivity D1000 – 5067–5584, ScreenTape; 5067–5585 Equipment Dounce homogenizer – 40 mL all glass homogenizer with large and small clearance pestles – Kimble Chase 885300–0040 Ultracentrifuge – Beckman Optima XE, Beckman SW41 Ti swinging rotor and buckets, Beckman Ultraclear 13.2 mL tubes (41121703) Refrigerated benchtop centrifuge similar to Eppendorf 5424R Nutrating Mixer FACS – BD FACSAria with 488, 561, 635/640 lasers Flow cytometry analyzer – BD LSR II with 488, 561, 640 lasers Software – Diva and FlowJo Agilent Bioanalyzer – with pico and nano chips for RNA Agilent TapeStation – D1000 and High Sensitivity D1000 screentapes and reagents Qubit Florometer Roche LightCycler 480 Covaris S220 Focused Ultrasonicator Dynamags from Thermo – 96 side magnet 12331D

    Affinity Purification:

    Article Title: Selective 2′-Hydroxyl Acylation analyzed by Protection from Exoribonuclease (RNase-detected SHAPE): direct analysis of covalent adducts and of nucleotide flexibility in RNA
    Article Snippet: .. M. genitalium RNase R, 4.5 mg/mL, conveniently obtained by affinity purification using a C-terminal (His)6 -tagged expression construct (see Supporting Protocol) Thiamine pyrophosphate (TPP) (Sigma-Aldrich, cat. no. C8754) Boric acid (Sigma-Aldrich, cat. no. B7901) SUPERase-In (Ambion, cat. no. AM2694) Alkaline phosphatase (Roche, cat. no. 1097075) T4 polynucleotide kinase (PNK) (New England Biolabs, cat. no. M0201S) 10× PNK buffer (New England Biolabs, cat. no. M0201S) γ-[32 P]-ATP (6 × 106 Ci/mol, 10 Ci/L, Perkin Elmer, cat. no. BLU502Z) Reagents for high-resolution polyacrylamide gel electrophoresis, including 29:1 acrylamide:bisacrylamide, 7 M urea, 1×TBE ..

    Binding Assay:

    Article Title: Novel insight into circular RNA HECTD1 in astrocyte activation via autophagy by targeting MIR142-TIPARP: implications for cerebral ischemic stroke
    Article Snippet: On the following day, the cells were transfected with 3ʹ-biotinylated MIR142 or MIR142 -mutation at a final concentration of 50 nM for 36 h. The cells were then washed with PBS, briefly vortexed, and incubated in lysis buffer (20 mM Tris, pH 7.5, 200 mM NaCl, 2.5 mM MgCl2 , 0.05% Igepal [Sigma-Aldrich, 18,896], 60 U/ml Superase-In [Invitrogen, AM2694], 1 mM DTT [Sigma-Aldrich, 43,816], and protease inhibitors [Biotool, ]) on ice for 10 min. Lysates were precleared by centrifugation, and 50-μl aliquots of the samples were prepared for input. .. The beads were coated with yeast tRNA in advance to prevent the nonspecific binding of RNA and protein complexes.

    DNA Extraction:

    Article Title: Identification of a transporter complex responsible for the cytosolic entry of nitrogen-containing bisphosphonates
    Article Snippet: .. In RNase Inhibitor (AM2694), SYBR Green qPCR master mix , Hygromycin B (10687010), DMEM (11965118), RPMI (11875093), Fetal Bovine Serum (FBS, 16000044) and Lipofectamine 3000 reagent (L3000008) were from ThermoFisher Scientific; HRP-conjugated anti-mouse and anti-rabbit secondary antibodies (1706515 and 1706516) were from Bio-Rad (Hercules, California); PNGase F (P0704) and Endo Hf (P0703) were from New England Biolabs (NEB, Ipswich, Massachusetts); cell line Nucleofector kit V (VACA-1003) was from Lonza (Walkersville, Maryland); CellTiter-Glo kit (G7572) was from Promega (Fitchburg, Wisconsin); mouse RANK ligand (RANKL, 462-TEC-010) was from R and D systems (Minneapolis, Minnesota); IMDM (30–2005) was from ATCC (American Type Culture Collection, Manassas, Virginia); digitonin (300410) was from Millipore-Sigma; QuickExtract DNA extraction solution (QE0905T) was from Epicentre (Madison, Wisconsin); tritium-labeled alendronate (MT-1727) was from Moravek (Brea, California); Alexa flour 647 labeled zoledronate (AF647-ZOL) was from BioVinc (Pasadena, California); Janelia flour 549 was a gift from Luke Lavis; pSpCas9(BB)−2A-GFP (PX458) was a gift from Feng Zhang (Addgene plasmid # 48138); AAVS1-Puro-PGK1 −3 × FLAG TwinStrep was a gift from Yannick Doyon (Addgene plasmid # 68375); pLenti PGK Hygro DEST (w530-1) was a gift from Eric Campeau and Paul Kaufman (Addgene plasmid # 19066); psPAX2 and pMD2.G were gifts from Didier Trono (Addgene plasmid # 12260 and # 12259). .. K562 human myeloid leukemia cells, Human Embryo Kidney (HEK) 293 T cells and RAW 264.7 murine macrophage-like cells were obtained from ATCC.

    RNA Sequencing Assay:

    Article Title: EINCR1 is an EGF inducible lincRNA overexpressed in lung adenocarcinomas
    Article Snippet: Paragraph title: Nuclear RNA-seq ... Briefly, cells were washed with 1xPBS and then incubated for 10 min with 1 ml of RLN buffer (50 mM TrisHCl pH = 8.0, 140 mM NaCl, 1.5 mM MgCl2 , 0.5% (v/v) IGEPAL CA-630 and 2 U/ml SUPERaseIn—RNase inhibitor, Invitrogen AM2696).

    Magnetic Beads:

    Article Title: Novel insight into circular RNA HECTD1 in astrocyte activation via autophagy by targeting MIR142-TIPARP: implications for cerebral ischemic stroke
    Article Snippet: On the following day, the cells were transfected with 3ʹ-biotinylated MIR142 or MIR142 -mutation at a final concentration of 50 nM for 36 h. The cells were then washed with PBS, briefly vortexed, and incubated in lysis buffer (20 mM Tris, pH 7.5, 200 mM NaCl, 2.5 mM MgCl2 , 0.05% Igepal [Sigma-Aldrich, 18,896], 60 U/ml Superase-In [Invitrogen, AM2694], 1 mM DTT [Sigma-Aldrich, 43,816], and protease inhibitors [Biotool, ]) on ice for 10 min. Lysates were precleared by centrifugation, and 50-μl aliquots of the samples were prepared for input. .. The remaining lysates were incubated with M-280 Streptavidin magnetic beads (Invitrogen, 11205D).

    Mutagenesis:

    Article Title: Novel insight into circular RNA HECTD1 in astrocyte activation via autophagy by targeting MIR142-TIPARP: implications for cerebral ischemic stroke
    Article Snippet: .. On the following day, the cells were transfected with 3ʹ-biotinylated MIR142 or MIR142 -mutation at a final concentration of 50 nM for 36 h. The cells were then washed with PBS, briefly vortexed, and incubated in lysis buffer (20 mM Tris, pH 7.5, 200 mM NaCl, 2.5 mM MgCl2 , 0.05% Igepal [Sigma-Aldrich, 18,896], 60 U/ml Superase-In [Invitrogen, AM2694], 1 mM DTT [Sigma-Aldrich, 43,816], and protease inhibitors [Biotool, ]) on ice for 10 min. Lysates were precleared by centrifugation, and 50-μl aliquots of the samples were prepared for input. .. The remaining lysates were incubated with M-280 Streptavidin magnetic beads (Invitrogen, 11205D).

    Isolation:

    Article Title: Nucleotide Resolution Comparison of Transcription of Human Cytomegalovirus and Host Genomes Reveals Universal Use of RNA Polymerase II Elongation Control Driven by Dissimilar Core Promoter Elements
    Article Snippet: All rapid nucleus isolation steps were performed on wet ice using ice-cold buffers as previously described ( ). .. After infection and treatment of cells, T150 flasks were removed from the incubator, and over 20 s, medium was discarded; phosphate-buffered saline (PBS) was added, quickly mixed, and discarded, and 10 ml lysis buffer (20 mM HEPES, pH 7.6, 300 mM sucrose, 1% IGEPAL CA-630, 1 mM spermine, 1 mM spermidine, 1 mM EDTA, 1 mM dithiothreitol [DTT], 0.004 U/μl SUPERase-In [Ambion AM2696], 0.1% isopropanol-saturated phenylmethylsulfonyl fluoride [PMSF], and cOmplete EDTA-free protease inhibitor cocktail [Roche 11873580001]) was introduced.

    Article Title: Chromatin run-on and sequencing maps the transcriptional regulatory landscape of glioblastoma multiforme
    Article Snippet: .. For chromatin (ChRO) isolation from cultured cells or tissue we added 1 ml of 1× NUN Buffer (0.3 M NaCl, 1M Urea, 1% NP-40, 20 mM HEPES, pH 7.5, 7.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 20 units/ml RNase Inhibitor (Life Technologies # AM2694), 1× Protease Inhibitor Cocktail (Roche # 11 873 580 001)). ..

    Article Title: Novel insight into circular RNA HECTD1 in astrocyte activation via autophagy by targeting MIR142-TIPARP: implications for cerebral ischemic stroke
    Article Snippet: Paragraph title: Affinity isolation assay with biotinylated miRNA ... On the following day, the cells were transfected with 3ʹ-biotinylated MIR142 or MIR142 -mutation at a final concentration of 50 nM for 36 h. The cells were then washed with PBS, briefly vortexed, and incubated in lysis buffer (20 mM Tris, pH 7.5, 200 mM NaCl, 2.5 mM MgCl2 , 0.05% Igepal [Sigma-Aldrich, 18,896], 60 U/ml Superase-In [Invitrogen, AM2694], 1 mM DTT [Sigma-Aldrich, 43,816], and protease inhibitors [Biotool, ]) on ice for 10 min. Lysates were precleared by centrifugation, and 50-μl aliquots of the samples were prepared for input.

    Article Title: Condensin controls recruitment of RNA polymerase II to achieve nematode X-chromosome dosage compensation
    Article Snippet: .. After washing twice with M9 buffer, animals were washed with cold nuclear isolation buffer (250 mM sucrose, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl2 , 1 mM EGTA, 0.25% NP-40, 1 mM DTT, protease inhibitors, 4 U/ml SUPERaseIn [AM2696; Ambion, Grand Island, NY]). .. Animals were resuspended in nuclear isolation buffer (embryos and starved L1 in 3 vol, L3 in 1 vol), and dripped into liquid nitrogen to freeze.

    Article Title: EINCR1 is an EGF inducible lincRNA overexpressed in lung adenocarcinomas
    Article Snippet: At the same time, nuclear fractions were isolated from cells which were not stimulated with EGF. .. Briefly, cells were washed with 1xPBS and then incubated for 10 min with 1 ml of RLN buffer (50 mM TrisHCl pH = 8.0, 140 mM NaCl, 1.5 mM MgCl2 , 0.5% (v/v) IGEPAL CA-630 and 2 U/ml SUPERaseIn—RNase inhibitor, Invitrogen AM2696).

    Flow Cytometry:

    Article Title: Species and cell-type properties of classically defined human and rodent neurons and glia
    Article Snippet: .. Other reagents Optiprep – Sigma D1556-250ML cOmplete mini protease inhibitors (EDTA free) – Roche distributed by Sigma 11836170001 Superasin – Thermo AM2696 RNasin – Promega N2515 16%formaldehyde solution – EMS 15710 BSA – Jackson 001-000-162 BSA RNase free – Thermo AM2618 DyeCycle Ruby – Thermo V10273 Kits Qiagen AllPrep FFPE – Cat #80234 Qiagen MinElute Reaction Cleanup – Cat #28206 Nugen Ovation RNAseq NEBNext Ultra II DNA Library Prep Kit for Illumina - E7645L NEBNext Multiplex Adapters for Illumina – E7335L (Set 1) and E7500L (Set 2) Qubit dsDNA High Sensitivity - Qubit RNA HS - Bioanalyzer Pico chips - 5067–1513 TapeStation D1000 - 5067–5582, ScreenTape; 5067–5583 TapeStation High Sensitivity D1000 – 5067–5584, ScreenTape; 5067–5585 Equipment Dounce homogenizer – 40 mL all glass homogenizer with large and small clearance pestles – Kimble Chase 885300–0040 Ultracentrifuge – Beckman Optima XE, Beckman SW41 Ti swinging rotor and buckets, Beckman Ultraclear 13.2 mL tubes (41121703) Refrigerated benchtop centrifuge similar to Eppendorf 5424R Nutrating Mixer FACS – BD FACSAria with 488, 561, 635/640 lasers Flow cytometry analyzer – BD LSR II with 488, 561, 640 lasers Software – Diva and FlowJo Agilent Bioanalyzer – with pico and nano chips for RNA Agilent TapeStation – D1000 and High Sensitivity D1000 screentapes and reagents Qubit Florometer Roche LightCycler 480 Covaris S220 Focused Ultrasonicator Dynamags from Thermo – 96 side magnet 12331D .. Mice were deeply anesthetized and transcardially perfused with phosphate buffered saline (PBS) followed by 4% formaldehyde (w/v) in PBS.

    Microscopy:

    Article Title: Nucleotide Resolution Comparison of Transcription of Human Cytomegalovirus and Host Genomes Reveals Universal Use of RNA Polymerase II Elongation Control Driven by Dissimilar Core Promoter Elements
    Article Snippet: After infection and treatment of cells, T150 flasks were removed from the incubator, and over 20 s, medium was discarded; phosphate-buffered saline (PBS) was added, quickly mixed, and discarded, and 10 ml lysis buffer (20 mM HEPES, pH 7.6, 300 mM sucrose, 1% IGEPAL CA-630, 1 mM spermine, 1 mM spermidine, 1 mM EDTA, 1 mM dithiothreitol [DTT], 0.004 U/μl SUPERase-In [Ambion AM2696], 0.1% isopropanol-saturated phenylmethylsulfonyl fluoride [PMSF], and cOmplete EDTA-free protease inhibitor cocktail [Roche 11873580001]) was introduced. .. After a brief incubation on ice, lysis was confirmed by phase-contrast microscopy.

    Cytometry:

    Article Title: Species and cell-type properties of classically defined human and rodent neurons and glia
    Article Snippet: .. Other reagents Optiprep – Sigma D1556-250ML cOmplete mini protease inhibitors (EDTA free) – Roche distributed by Sigma 11836170001 Superasin – Thermo AM2696 RNasin – Promega N2515 16%formaldehyde solution – EMS 15710 BSA – Jackson 001-000-162 BSA RNase free – Thermo AM2618 DyeCycle Ruby – Thermo V10273 Kits Qiagen AllPrep FFPE – Cat #80234 Qiagen MinElute Reaction Cleanup – Cat #28206 Nugen Ovation RNAseq NEBNext Ultra II DNA Library Prep Kit for Illumina - E7645L NEBNext Multiplex Adapters for Illumina – E7335L (Set 1) and E7500L (Set 2) Qubit dsDNA High Sensitivity - Qubit RNA HS - Bioanalyzer Pico chips - 5067–1513 TapeStation D1000 - 5067–5582, ScreenTape; 5067–5583 TapeStation High Sensitivity D1000 – 5067–5584, ScreenTape; 5067–5585 Equipment Dounce homogenizer – 40 mL all glass homogenizer with large and small clearance pestles – Kimble Chase 885300–0040 Ultracentrifuge – Beckman Optima XE, Beckman SW41 Ti swinging rotor and buckets, Beckman Ultraclear 13.2 mL tubes (41121703) Refrigerated benchtop centrifuge similar to Eppendorf 5424R Nutrating Mixer FACS – BD FACSAria with 488, 561, 635/640 lasers Flow cytometry analyzer – BD LSR II with 488, 561, 640 lasers Software – Diva and FlowJo Agilent Bioanalyzer – with pico and nano chips for RNA Agilent TapeStation – D1000 and High Sensitivity D1000 screentapes and reagents Qubit Florometer Roche LightCycler 480 Covaris S220 Focused Ultrasonicator Dynamags from Thermo – 96 side magnet 12331D .. Mice were deeply anesthetized and transcardially perfused with phosphate buffered saline (PBS) followed by 4% formaldehyde (w/v) in PBS.

    Quantitative RT-PCR:

    Article Title: Convergence of independent DISC1 mutations on impaired neurite growth via decreased UNC5D expression
    Article Snippet: Paragraph title: Single-cell qRT-PCR ... Day 28 cultures were single-cell sorted for GFP expression (marking transduced neurons) into lysis buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA with 0.5% NP40 [Thermo Scientific PI-28324] and 100 U/ml SUPERase•In™ [Ambion AM2696]).

    Polyacrylamide Gel Electrophoresis:

    Article Title: Selective 2′-Hydroxyl Acylation analyzed by Protection from Exoribonuclease (RNase-detected SHAPE): direct analysis of covalent adducts and of nucleotide flexibility in RNA
    Article Snippet: .. M. genitalium RNase R, 4.5 mg/mL, conveniently obtained by affinity purification using a C-terminal (His)6 -tagged expression construct (see Supporting Protocol) Thiamine pyrophosphate (TPP) (Sigma-Aldrich, cat. no. C8754) Boric acid (Sigma-Aldrich, cat. no. B7901) SUPERase-In (Ambion, cat. no. AM2694) Alkaline phosphatase (Roche, cat. no. 1097075) T4 polynucleotide kinase (PNK) (New England Biolabs, cat. no. M0201S) 10× PNK buffer (New England Biolabs, cat. no. M0201S) γ-[32 P]-ATP (6 × 106 Ci/mol, 10 Ci/L, Perkin Elmer, cat. no. BLU502Z) Reagents for high-resolution polyacrylamide gel electrophoresis, including 29:1 acrylamide:bisacrylamide, 7 M urea, 1×TBE ..

    Lysis:

    Article Title: Nucleotide Resolution Comparison of Transcription of Human Cytomegalovirus and Host Genomes Reveals Universal Use of RNA Polymerase II Elongation Control Driven by Dissimilar Core Promoter Elements
    Article Snippet: .. After infection and treatment of cells, T150 flasks were removed from the incubator, and over 20 s, medium was discarded; phosphate-buffered saline (PBS) was added, quickly mixed, and discarded, and 10 ml lysis buffer (20 mM HEPES, pH 7.6, 300 mM sucrose, 1% IGEPAL CA-630, 1 mM spermine, 1 mM spermidine, 1 mM EDTA, 1 mM dithiothreitol [DTT], 0.004 U/μl SUPERase-In [Ambion AM2696], 0.1% isopropanol-saturated phenylmethylsulfonyl fluoride [PMSF], and cOmplete EDTA-free protease inhibitor cocktail [Roche 11873580001]) was introduced. .. After a brief incubation on ice, lysis was confirmed by phase-contrast microscopy.

    Article Title: Enhanced Replication of Mouse Adenovirus Type 1 following Virus-Induced Degradation of Protein Kinase R (PKR)
    Article Snippet: .. Cells were collected at 24 hpi by scraping in ice-cold PBS containing 100 µg/ml cycloheximide, pelleting, and resuspending in 500 µl lysis buffer (20 mM Tris-Cl, 150 mM NaCl, 15 mM MgCl2 , 8% glycerol, 20 IU/ml SUPERase•In [Thermo Fisher Scientific catalog no. AM2696], 80 IU/ml murine RNase inhibitor [New England BioLabs catalog no. M0314S], 0.1 mg/ml heparin [Sigma H3393-50], 0.1 mg/ml cycloheximide, 1 mM DTT, 1× protease inhibitor [protease inhibitor cocktail kit; Thermo Scientific catalog no. 78410], 20 IU/ml Turbo DNase [Thermo Fisher Scientific catalog no. AM2238], 1% Triton X-100 [Sigma T9284]). .. Cells were lysed by passage through a chilled 26-gauge needle 10 times, vortex mixing for 30 s, and then incubating on ice for 5 min. Lysates were cleared by centrifugation for 5 min at 14,000 × g at 4°C.

    Article Title: Novel insight into circular RNA HECTD1 in astrocyte activation via autophagy by targeting MIR142-TIPARP: implications for cerebral ischemic stroke
    Article Snippet: .. On the following day, the cells were transfected with 3ʹ-biotinylated MIR142 or MIR142 -mutation at a final concentration of 50 nM for 36 h. The cells were then washed with PBS, briefly vortexed, and incubated in lysis buffer (20 mM Tris, pH 7.5, 200 mM NaCl, 2.5 mM MgCl2 , 0.05% Igepal [Sigma-Aldrich, 18,896], 60 U/ml Superase-In [Invitrogen, AM2694], 1 mM DTT [Sigma-Aldrich, 43,816], and protease inhibitors [Biotool, ]) on ice for 10 min. Lysates were precleared by centrifugation, and 50-μl aliquots of the samples were prepared for input. .. The remaining lysates were incubated with M-280 Streptavidin magnetic beads (Invitrogen, 11205D).

    Article Title: Combining NGN2 Programming with Developmental Patterning Generates Human Excitatory Neurons with NMDAR-Mediated Synaptic Transmission
    Article Snippet: Cells were sorted by BD Facs Aria, 20 psi, 40–100 ev/s into 96-well plates (one cell per well) containing 5 μL lysis buffer. .. For Fluidigm Biomark, cells were single-cell sorted into 5 μL of 10 mM Tris (pH 8.0), 0.1 mM EDTA supplemented with SUPERase•In (0.1 U/μL final, Ambion, AM2696), and 0.5% NP40 (Thermo Scientific, PI-28324).

    Article Title: Convergence of independent DISC1 mutations on impaired neurite growth via decreased UNC5D expression
    Article Snippet: .. Day 28 cultures were single-cell sorted for GFP expression (marking transduced neurons) into lysis buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA with 0.5% NP40 [Thermo Scientific PI-28324] and 100 U/ml SUPERase•In™ [Ambion AM2696]). ..

    Article Title: Ras Suppresses TXNIP Expression by Restricting Ribosome Translocation
    Article Snippet: .. The cell pellet was collected and lysed in polysome lysis buffer (15 mM Tris [pH 7.4], 15 mM MgCl2 , 100 mM KCl, 1% Triton X-100, 1 mM DTT, 100 μg/ml CHX, 500 U/ml SuperaseIn [Invitrogen; no. AM2696]) containing protease inhibitors (1 mM PMSF, 2.5 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin) and phosphatase inhibitors (phosphatase inhibitor cocktails 1 and 2; Sigma; no. P2850 and P5726). .. The cell lysate was loaded onto the top of a sucrose gradient and centrifuged at 47,000 rpm for 1 h at 4°C in a SW55 Ti rotor (Beckman; no. 342194), and centrifugation was stopped with no brake.

    Plasmid Preparation:

    Article Title: Identification of a transporter complex responsible for the cytosolic entry of nitrogen-containing bisphosphonates
    Article Snippet: .. In RNase Inhibitor (AM2694), SYBR Green qPCR master mix , Hygromycin B (10687010), DMEM (11965118), RPMI (11875093), Fetal Bovine Serum (FBS, 16000044) and Lipofectamine 3000 reagent (L3000008) were from ThermoFisher Scientific; HRP-conjugated anti-mouse and anti-rabbit secondary antibodies (1706515 and 1706516) were from Bio-Rad (Hercules, California); PNGase F (P0704) and Endo Hf (P0703) were from New England Biolabs (NEB, Ipswich, Massachusetts); cell line Nucleofector kit V (VACA-1003) was from Lonza (Walkersville, Maryland); CellTiter-Glo kit (G7572) was from Promega (Fitchburg, Wisconsin); mouse RANK ligand (RANKL, 462-TEC-010) was from R and D systems (Minneapolis, Minnesota); IMDM (30–2005) was from ATCC (American Type Culture Collection, Manassas, Virginia); digitonin (300410) was from Millipore-Sigma; QuickExtract DNA extraction solution (QE0905T) was from Epicentre (Madison, Wisconsin); tritium-labeled alendronate (MT-1727) was from Moravek (Brea, California); Alexa flour 647 labeled zoledronate (AF647-ZOL) was from BioVinc (Pasadena, California); Janelia flour 549 was a gift from Luke Lavis; pSpCas9(BB)−2A-GFP (PX458) was a gift from Feng Zhang (Addgene plasmid # 48138); AAVS1-Puro-PGK1 −3 × FLAG TwinStrep was a gift from Yannick Doyon (Addgene plasmid # 68375); pLenti PGK Hygro DEST (w530-1) was a gift from Eric Campeau and Paul Kaufman (Addgene plasmid # 19066); psPAX2 and pMD2.G were gifts from Didier Trono (Addgene plasmid # 12260 and # 12259). .. K562 human myeloid leukemia cells, Human Embryo Kidney (HEK) 293 T cells and RAW 264.7 murine macrophage-like cells were obtained from ATCC.

    Software:

    Article Title: Species and cell-type properties of classically defined human and rodent neurons and glia
    Article Snippet: .. Other reagents Optiprep – Sigma D1556-250ML cOmplete mini protease inhibitors (EDTA free) – Roche distributed by Sigma 11836170001 Superasin – Thermo AM2696 RNasin – Promega N2515 16%formaldehyde solution – EMS 15710 BSA – Jackson 001-000-162 BSA RNase free – Thermo AM2618 DyeCycle Ruby – Thermo V10273 Kits Qiagen AllPrep FFPE – Cat #80234 Qiagen MinElute Reaction Cleanup – Cat #28206 Nugen Ovation RNAseq NEBNext Ultra II DNA Library Prep Kit for Illumina - E7645L NEBNext Multiplex Adapters for Illumina – E7335L (Set 1) and E7500L (Set 2) Qubit dsDNA High Sensitivity - Qubit RNA HS - Bioanalyzer Pico chips - 5067–1513 TapeStation D1000 - 5067–5582, ScreenTape; 5067–5583 TapeStation High Sensitivity D1000 – 5067–5584, ScreenTape; 5067–5585 Equipment Dounce homogenizer – 40 mL all glass homogenizer with large and small clearance pestles – Kimble Chase 885300–0040 Ultracentrifuge – Beckman Optima XE, Beckman SW41 Ti swinging rotor and buckets, Beckman Ultraclear 13.2 mL tubes (41121703) Refrigerated benchtop centrifuge similar to Eppendorf 5424R Nutrating Mixer FACS – BD FACSAria with 488, 561, 635/640 lasers Flow cytometry analyzer – BD LSR II with 488, 561, 640 lasers Software – Diva and FlowJo Agilent Bioanalyzer – with pico and nano chips for RNA Agilent TapeStation – D1000 and High Sensitivity D1000 screentapes and reagents Qubit Florometer Roche LightCycler 480 Covaris S220 Focused Ultrasonicator Dynamags from Thermo – 96 side magnet 12331D .. Mice were deeply anesthetized and transcardially perfused with phosphate buffered saline (PBS) followed by 4% formaldehyde (w/v) in PBS.

    SYBR Green Assay:

    Article Title: Identification of a transporter complex responsible for the cytosolic entry of nitrogen-containing bisphosphonates
    Article Snippet: .. In RNase Inhibitor (AM2694), SYBR Green qPCR master mix , Hygromycin B (10687010), DMEM (11965118), RPMI (11875093), Fetal Bovine Serum (FBS, 16000044) and Lipofectamine 3000 reagent (L3000008) were from ThermoFisher Scientific; HRP-conjugated anti-mouse and anti-rabbit secondary antibodies (1706515 and 1706516) were from Bio-Rad (Hercules, California); PNGase F (P0704) and Endo Hf (P0703) were from New England Biolabs (NEB, Ipswich, Massachusetts); cell line Nucleofector kit V (VACA-1003) was from Lonza (Walkersville, Maryland); CellTiter-Glo kit (G7572) was from Promega (Fitchburg, Wisconsin); mouse RANK ligand (RANKL, 462-TEC-010) was from R and D systems (Minneapolis, Minnesota); IMDM (30–2005) was from ATCC (American Type Culture Collection, Manassas, Virginia); digitonin (300410) was from Millipore-Sigma; QuickExtract DNA extraction solution (QE0905T) was from Epicentre (Madison, Wisconsin); tritium-labeled alendronate (MT-1727) was from Moravek (Brea, California); Alexa flour 647 labeled zoledronate (AF647-ZOL) was from BioVinc (Pasadena, California); Janelia flour 549 was a gift from Luke Lavis; pSpCas9(BB)−2A-GFP (PX458) was a gift from Feng Zhang (Addgene plasmid # 48138); AAVS1-Puro-PGK1 −3 × FLAG TwinStrep was a gift from Yannick Doyon (Addgene plasmid # 68375); pLenti PGK Hygro DEST (w530-1) was a gift from Eric Campeau and Paul Kaufman (Addgene plasmid # 19066); psPAX2 and pMD2.G were gifts from Didier Trono (Addgene plasmid # 12260 and # 12259). .. K562 human myeloid leukemia cells, Human Embryo Kidney (HEK) 293 T cells and RAW 264.7 murine macrophage-like cells were obtained from ATCC.

    Multiplex Assay:

    Article Title: Species and cell-type properties of classically defined human and rodent neurons and glia
    Article Snippet: .. Other reagents Optiprep – Sigma D1556-250ML cOmplete mini protease inhibitors (EDTA free) – Roche distributed by Sigma 11836170001 Superasin – Thermo AM2696 RNasin – Promega N2515 16%formaldehyde solution – EMS 15710 BSA – Jackson 001-000-162 BSA RNase free – Thermo AM2618 DyeCycle Ruby – Thermo V10273 Kits Qiagen AllPrep FFPE – Cat #80234 Qiagen MinElute Reaction Cleanup – Cat #28206 Nugen Ovation RNAseq NEBNext Ultra II DNA Library Prep Kit for Illumina - E7645L NEBNext Multiplex Adapters for Illumina – E7335L (Set 1) and E7500L (Set 2) Qubit dsDNA High Sensitivity - Qubit RNA HS - Bioanalyzer Pico chips - 5067–1513 TapeStation D1000 - 5067–5582, ScreenTape; 5067–5583 TapeStation High Sensitivity D1000 – 5067–5584, ScreenTape; 5067–5585 Equipment Dounce homogenizer – 40 mL all glass homogenizer with large and small clearance pestles – Kimble Chase 885300–0040 Ultracentrifuge – Beckman Optima XE, Beckman SW41 Ti swinging rotor and buckets, Beckman Ultraclear 13.2 mL tubes (41121703) Refrigerated benchtop centrifuge similar to Eppendorf 5424R Nutrating Mixer FACS – BD FACSAria with 488, 561, 635/640 lasers Flow cytometry analyzer – BD LSR II with 488, 561, 640 lasers Software – Diva and FlowJo Agilent Bioanalyzer – with pico and nano chips for RNA Agilent TapeStation – D1000 and High Sensitivity D1000 screentapes and reagents Qubit Florometer Roche LightCycler 480 Covaris S220 Focused Ultrasonicator Dynamags from Thermo – 96 side magnet 12331D .. Mice were deeply anesthetized and transcardially perfused with phosphate buffered saline (PBS) followed by 4% formaldehyde (w/v) in PBS.

    Concentration Assay:

    Article Title: Selective 2′-Hydroxyl Acylation analyzed by Protection from Exoribonuclease (RNase-detected SHAPE): direct analysis of covalent adducts and of nucleotide flexibility in RNA
    Article Snippet: 5′-[32 P]-labeled RNA at a concentration of 0.1 μM or greater in 10 mM HEPES, pH 8.0 CRITICAL: RNA preparation must be performed in an RNase-free environment. .. M. genitalium RNase R, 4.5 mg/mL, conveniently obtained by affinity purification using a C-terminal (His)6 -tagged expression construct (see Supporting Protocol) Thiamine pyrophosphate (TPP) (Sigma-Aldrich, cat. no. C8754) Boric acid (Sigma-Aldrich, cat. no. B7901) SUPERase-In (Ambion, cat. no. AM2694) Alkaline phosphatase (Roche, cat. no. 1097075) T4 polynucleotide kinase (PNK) (New England Biolabs, cat. no. M0201S) 10× PNK buffer (New England Biolabs, cat. no. M0201S) γ-[32 P]-ATP (6 × 106 Ci/mol, 10 Ci/L, Perkin Elmer, cat. no. BLU502Z) Reagents for high-resolution polyacrylamide gel electrophoresis, including 29:1 acrylamide:bisacrylamide, 7 M urea, 1×TBE

    Article Title: Novel insight into circular RNA HECTD1 in astrocyte activation via autophagy by targeting MIR142-TIPARP: implications for cerebral ischemic stroke
    Article Snippet: .. On the following day, the cells were transfected with 3ʹ-biotinylated MIR142 or MIR142 -mutation at a final concentration of 50 nM for 36 h. The cells were then washed with PBS, briefly vortexed, and incubated in lysis buffer (20 mM Tris, pH 7.5, 200 mM NaCl, 2.5 mM MgCl2 , 0.05% Igepal [Sigma-Aldrich, 18,896], 60 U/ml Superase-In [Invitrogen, AM2694], 1 mM DTT [Sigma-Aldrich, 43,816], and protease inhibitors [Biotool, ]) on ice for 10 min. Lysates were precleared by centrifugation, and 50-μl aliquots of the samples were prepared for input. .. The remaining lysates were incubated with M-280 Streptavidin magnetic beads (Invitrogen, 11205D).

    Article Title: Species and cell-type properties of classically defined human and rodent neurons and glia
    Article Snippet: Chemicals from Sigma Sucrose, Tricine, spermidine trihydrochloride, spermine tetrahydrochloride, DTT, Tris, NaCl, KCl, MgCl2 , Glycerol, TritonX-100, NP-40 Make stock solution to concentration indicated. .. Other reagents Optiprep – Sigma D1556-250ML cOmplete mini protease inhibitors (EDTA free) – Roche distributed by Sigma 11836170001 Superasin – Thermo AM2696 RNasin – Promega N2515 16%formaldehyde solution – EMS 15710 BSA – Jackson 001-000-162 BSA RNase free – Thermo AM2618 DyeCycle Ruby – Thermo V10273 Kits Qiagen AllPrep FFPE – Cat #80234 Qiagen MinElute Reaction Cleanup – Cat #28206 Nugen Ovation RNAseq NEBNext Ultra II DNA Library Prep Kit for Illumina - E7645L NEBNext Multiplex Adapters for Illumina – E7335L (Set 1) and E7500L (Set 2) Qubit dsDNA High Sensitivity - Qubit RNA HS - Bioanalyzer Pico chips - 5067–1513 TapeStation D1000 - 5067–5582, ScreenTape; 5067–5583 TapeStation High Sensitivity D1000 – 5067–5584, ScreenTape; 5067–5585 Equipment Dounce homogenizer – 40 mL all glass homogenizer with large and small clearance pestles – Kimble Chase 885300–0040 Ultracentrifuge – Beckman Optima XE, Beckman SW41 Ti swinging rotor and buckets, Beckman Ultraclear 13.2 mL tubes (41121703) Refrigerated benchtop centrifuge similar to Eppendorf 5424R Nutrating Mixer FACS – BD FACSAria with 488, 561, 635/640 lasers Flow cytometry analyzer – BD LSR II with 488, 561, 640 lasers Software – Diva and FlowJo Agilent Bioanalyzer – with pico and nano chips for RNA Agilent TapeStation – D1000 and High Sensitivity D1000 screentapes and reagents Qubit Florometer Roche LightCycler 480 Covaris S220 Focused Ultrasonicator Dynamags from Thermo – 96 side magnet 12331D

    Article Title: Using intracellular markers to identify a novel set of surface markers for live cell purification from a heterogeneous hIPSC culture
    Article Snippet: Next, the cell pellet was resuspended in fresh 4% paraformaldehyde (made in RNAse-free PBS) at a concentration of 1 mL per 5 × 106 cells and incubated at room temperature for 15 minutes. .. The washed pellet was then resuspended in perm/block buffer (0.5% saponin, 1% BSA, 200 U/mL Superase.In RNAse inhibitor (Life Technologies cat# AM2696), in RNAse-free PBS), in the same volume as used for fixation, with primary antibody and incubated at room temperature for two hours with occasional mixing.

    Article Title: EINCR1 is an EGF inducible lincRNA overexpressed in lung adenocarcinomas
    Article Snippet: After 48 hrs incubation, EGF was added to a final concentration of 20 ng/ml. .. Briefly, cells were washed with 1xPBS and then incubated for 10 min with 1 ml of RLN buffer (50 mM TrisHCl pH = 8.0, 140 mM NaCl, 1.5 mM MgCl2 , 0.5% (v/v) IGEPAL CA-630 and 2 U/ml SUPERaseIn—RNase inhibitor, Invitrogen AM2696).

    Fractionation:

    Article Title: Enhanced Replication of Mouse Adenovirus Type 1 following Virus-Induced Degradation of Protein Kinase R (PKR)
    Article Snippet: Cells were collected at 24 hpi by scraping in ice-cold PBS containing 100 µg/ml cycloheximide, pelleting, and resuspending in 500 µl lysis buffer (20 mM Tris-Cl, 150 mM NaCl, 15 mM MgCl2 , 8% glycerol, 20 IU/ml SUPERase•In [Thermo Fisher Scientific catalog no. AM2696], 80 IU/ml murine RNase inhibitor [New England BioLabs catalog no. M0314S], 0.1 mg/ml heparin [Sigma H3393-50], 0.1 mg/ml cycloheximide, 1 mM DTT, 1× protease inhibitor [protease inhibitor cocktail kit; Thermo Scientific catalog no. 78410], 20 IU/ml Turbo DNase [Thermo Fisher Scientific catalog no. AM2238], 1% Triton X-100 [Sigma T9284]). .. After centrifugation, gradients were pumped out of the top with a Brandel BR-188 density gradient fractionation system with a continuous reading of the OD254 .

    Article Title: Ras Suppresses TXNIP Expression by Restricting Ribosome Translocation
    Article Snippet: The cell pellet was collected and lysed in polysome lysis buffer (15 mM Tris [pH 7.4], 15 mM MgCl2 , 100 mM KCl, 1% Triton X-100, 1 mM DTT, 100 μg/ml CHX, 500 U/ml SuperaseIn [Invitrogen; no. AM2696]) containing protease inhibitors (1 mM PMSF, 2.5 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin) and phosphatase inhibitors (phosphatase inhibitor cocktails 1 and 2; Sigma; no. P2850 and P5726). .. After fractionation, fractions corresponding to 40S, 60S, and 80S peaks were combined, and fraction numbers were assigned ( and show the fraction numbers on the x axis).

    FACS:

    Article Title: Species and cell-type properties of classically defined human and rodent neurons and glia
    Article Snippet: .. Other reagents Optiprep – Sigma D1556-250ML cOmplete mini protease inhibitors (EDTA free) – Roche distributed by Sigma 11836170001 Superasin – Thermo AM2696 RNasin – Promega N2515 16%formaldehyde solution – EMS 15710 BSA – Jackson 001-000-162 BSA RNase free – Thermo AM2618 DyeCycle Ruby – Thermo V10273 Kits Qiagen AllPrep FFPE – Cat #80234 Qiagen MinElute Reaction Cleanup – Cat #28206 Nugen Ovation RNAseq NEBNext Ultra II DNA Library Prep Kit for Illumina - E7645L NEBNext Multiplex Adapters for Illumina – E7335L (Set 1) and E7500L (Set 2) Qubit dsDNA High Sensitivity - Qubit RNA HS - Bioanalyzer Pico chips - 5067–1513 TapeStation D1000 - 5067–5582, ScreenTape; 5067–5583 TapeStation High Sensitivity D1000 – 5067–5584, ScreenTape; 5067–5585 Equipment Dounce homogenizer – 40 mL all glass homogenizer with large and small clearance pestles – Kimble Chase 885300–0040 Ultracentrifuge – Beckman Optima XE, Beckman SW41 Ti swinging rotor and buckets, Beckman Ultraclear 13.2 mL tubes (41121703) Refrigerated benchtop centrifuge similar to Eppendorf 5424R Nutrating Mixer FACS – BD FACSAria with 488, 561, 635/640 lasers Flow cytometry analyzer – BD LSR II with 488, 561, 640 lasers Software – Diva and FlowJo Agilent Bioanalyzer – with pico and nano chips for RNA Agilent TapeStation – D1000 and High Sensitivity D1000 screentapes and reagents Qubit Florometer Roche LightCycler 480 Covaris S220 Focused Ultrasonicator Dynamags from Thermo – 96 side magnet 12331D .. Mice were deeply anesthetized and transcardially perfused with phosphate buffered saline (PBS) followed by 4% formaldehyde (w/v) in PBS.

    Article Title: Combining NGN2 Programming with Developmental Patterning Generates Human Excitatory Neurons with NMDAR-Mediated Synaptic Transmission
    Article Snippet: Cells were sorted by BD Facs Aria, 20 psi, 40–100 ev/s into 96-well plates (one cell per well) containing 5 μL lysis buffer. .. For Fluidigm Biomark, cells were single-cell sorted into 5 μL of 10 mM Tris (pH 8.0), 0.1 mM EDTA supplemented with SUPERase•In (0.1 U/μL final, Ambion, AM2696), and 0.5% NP40 (Thermo Scientific, PI-28324).

    Single-cell Analysis:

    Article Title: Convergence of independent DISC1 mutations on impaired neurite growth via decreased UNC5D expression
    Article Snippet: WTex8 lines A & B and MUTex8 lines A & D were used for this single-cell analysis. .. Day 28 cultures were single-cell sorted for GFP expression (marking transduced neurons) into lysis buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA with 0.5% NP40 [Thermo Scientific PI-28324] and 100 U/ml SUPERase•In™ [Ambion AM2696]).

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    Thermo Fisher 11836170001 superasin thermo am2696 rnasin promega n2515
    11836170001 Superasin Thermo Am2696 Rnasin Promega N2515, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 2 article reviews
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    11836170001 superasin thermo am2696 rnasin promega n2515 - by Bioz Stars, 2020-02
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